SOLID-STATE 2D-NMR INVESTIGATION OF DNA & ITS COMPLEXES

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We propose to apply a two-dimensional solid-state NMR technique, recently invented by the PI, to the determination of the structure of DNA and of bound molecules associated with it. The technique uses magic-angle spinning to acquire high- resolution NMR spectra of oriented DNA films produced by a fiber-spinning technique, while encoding the orientations of each group in the polymer as sideband intensities in the 2D spectra. These intensities can be directly used to give the orientational distribution function of individual groups: without prior assumptions these functions can be combined to generate the structure of the molecule. The proposed technique has some advantages over existing physical methods of structural determination, and because it probes molecular orientations rather than dimensions, is likely to be complementary to them. Preliminary calculations show that the spectra distinguish between the major conformations of DNA, between different models of the structure of these conformations, and between different alignments of bound drug molecules relative to the DNA fiber. We propose first of all to construct an apparatus for the reproducible production of films of oriented native DNA in the various forms which can be produced by altering electrolyte concentration and ambient humidity, and to analyses via 2D-NMR the structure of these conformers, refining and removing ambiguities in existing models. We shall then examine the conformation of synthetic DNAs of defined sequence, and compare their structures with those of native materials. Finally, we shall synthesize a series of isotopically-labelled DNA-binding ligands, among them several clinically important cytotoxic and antiviral drugs, and determine the structure of their complexes with high-molecular-weight DNA. This will be possible at very low ratios of drug molecules to DNA base pairs. The ultimate goal is to establish a new physico-chemical tool to aid in the characterization and development of novel pharmacological materials, and to add to the understanding of the processes of genetic replication, transcription and regulation.